Thionucleotides in mammalian tRNA are synthesized at the polynucleotide level, by the transfer of sulfur from cysteine to acceptor sites in the newly synthesized tRNA molecule. We have characterized the rat liver enzyme which catalyzes this reaction. We also found that while DAB-induced hepatomas contain a similar tRNA sulfurtransferase activity, differences existed in specific thiobases produced by the liver and tumor enzymes. We propose to further investigate these enzymes using several approaches. First, the enzymes will be purified from rat liver and then hepatomas, to free them of enzymes which compete for cysteine in our assay system. Hopefully, the activity will become stable at some stage of purification, thus facilitating later work. Secondly, sulfurtransferases will be purified from other tumors to gain a more general understanding of the state of these enzymes in cancer tissues. Finally, the isolated rat hepatocyte system is being developed to study thionucleotide synthesis in intact cells. This system offers the advantages of providing many cells of one cell type, and allows several variables to be manipulated using cells from a single liver. We have shown that the thionucleotides from hepatocytes are the same as those produced by tRNA sulfurtransferase. Hence, we should be able to use the hepatocyte system to identify the in vitro thionucleotides. The goal of these studies is to learn the relationship between thionucleotide synthesis and growth, both normal and abnormal. BIBLIOGRAPHIC REFERENCES: RNA Synthesis and Maturation in Isolated Rat Hepatocytes, G. G. Harmison, II, J. B. Blair, and C. L. Harris. Federation Proceedings 35, 1511 (1976). Isoleucyl-tRNA Synthetase Levels and the Extent of Aminoacylation of tRNAile from E. coli. F. Marashi and C. L. Harris. Federation Proceedings 35, 1467 (1976).